Method for selection of primate tumor-associated antigens suitable as in vivo targets for antibodies

ABSTRACT

The present invention is a process for the selection of antigens which are suitable targets for in vivo antibody localization in human tumors or other altered (or diseased) tissue. The process provides a simplified and rapid technique for discovering useful in vivo targets for antibodies and is useful in cancer detection and therapy in humans or other primates, whether or not the antigens are specific to tumors. More specifically, the invention relates to a process for the selection of antigens suitable as targets for antibodies which localize in a tumor in vivo in which antibodies are first prepared distinguishable from those present in the animal in which biofiltration is to occur and that bind to antigens present in the tumor to be targeted. These antibodies are then injected into a non-tumor-bearing primate, into a tumor-bearing animal, and into a non-tumor bearing animal of the same species as the tumor-bearing animal to permit biofiltration of the antibodies. The biofiltered antibodies are next recovered from each of the non-tumor-bearing primate, the tumor-bearing animal, and the non-tumor-bearing animal and are employed to identify antigens whose antibodies are not retained in vivo in the primate and the non-tumor-bearing animal. The antibodies that are not retained in vivo by the non-tumor-bearing primate and the non-tumor bearing animal are then compared with those antibodies that are actually retained in vivo in the tumor-bearing animal to identify the antigens corresponding to those antibodies that are selectively retained in the tumor-bearing animal. In an alternative process, the tumor-bearing animal may be replaced by a perfusable surgically removed human organ bearing a tumor, and the non-tumor-bearing animal may be replaced by a surgically removed normal organ. Passage through a primate, however, remains the method of choice for selecting antigens whose antibodies are unabsorbed in vivo.

ACKNOWLEDGEMENT

The invention described herein was made in part with the use ofintramural research funds from the United States VeteransAdministration.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of copending application(s) Ser. No. 07/295,775filed on 1/11/89, now U.S. Pat. No. 4,978,520, which in turn is acontinuation-in-part of U.S. Pat. application Ser. No. 06/906,161, filedSept. 11, 1986, now U.S. Pat. No. 4,798,719.

FIELD OF THE INVENTION

The present invention relates to a process for the selection ofantigenic targets which can serve for selective in vivo localization oftumor-localizing antibodies in primates, including humans.

BACKGROUND OF THE INVENTION

The use of appropriately labeled antibodies for tumor location has beensuggested in the literature for many years (D. Pressman and G. Keighley,J. Immunology 59, 141 (1948); reviewed in D. Pressman, Handbook ofCancer Immunology, H. Waters Ed. (Garland STPM, New York, 1978) Vol. 5,pp. 29-50). However, the difficulty of obtaining reproducible,tumor-specific antibodies has markedly hindered advances in this area.

It has been demonstrated that monoclonal antibodies can give improvedtumor location, partly because of the absence of interferingnon-specific antibodies, and partly because of the high specificity andabsence of cross-reactivity available in monoclonal antibodies (Ballouet al., Science 206, 844 (1979)). However, successful targeting usingmonoclonal antibodies in animals and humans has generally beendisappointing. It is believed that the principal reason for suchdisappointing results has been the generally accepted assumption thatmonoclonal antibodies which are specific to tumors, and not to normaltissue, are required. Producing such antibodies has proved-difficult andfew, if any, have been found in spite of extensive researchinvestigators in the area.

It has recently been demonstrated that absolute specificity to tumors isnot necessary for proper tumor location (Ballou et al., J. Immunology132, 2111-2116 (1984)). In that publication, a monoclonal antibody to atarget present at much higher levels in normal tissues than in tumorswas shown to localize specifically in tumors and not in the antigenicnormal tissues.

In vitro and in vivo specificity are quite different as indicated by twodifferent findings:

First, monoclonal antibodies which localize in tumors need not be trulytumor specific: examples of such antibodies are A2B5 (Reintgen et al.,J. Surg. Oncol. 23, 205-211 (1983)) and anti-SSEA-1 (Ballou et al., J.Immunology, 132, 2111-2116 (1984)).

Second, antibodies which show a high level of in vitro specificity maynot localize effectively. Mann et al., (Cancer 54, 1318 (1984))discloses monoclonal antibodies having an apparent specificity for eachof two human tumors. The expectation was that, when the tumors wereimplanted in a living animal, and the antibodies were radiolabeleddifferently so as to enable them to be distinguished from each other,then each antibody would go only to the appropriate tumor. The results,however, showed that neither antibody localized to any appreciableextent.

Thus antibodies which target tumors selectively need not be specific,and antibodies which are apparently specific may not target.

In spite of these findings, tumor-localizing antibodies have beenprepared by selecting monoclonal antibodies that are more highly boundto tumors than to normal tissues in in vitro screening assays. Thepresent invention is for a novel selection methodology, which permitsanalysis of a wider range of antigens than are selectable by in vitroscreening.

The present invention was developed to permit selection of antigens thatare selectively targeted in tumors of primates, including humans, bybiofiltration methodology, while avoiding the ethically dubiousinjection of antibodies to human tumors into humans.

SUMMARY OF THE INVENTION

The subject invention is a process for the selection of antigenssuitable as targets for antibodies which localize in a tumor in vivocomprising:

(a) preparing antibodies distinguishable from those present in theanimal in which biofiltration is to occur and that bind to antigenspresent in the tumor to be targeted;

(b) injecting the prepared antibodies into a non-tumor-bearing primate,a tumor-bearing animal, and a non-tumor bearing animal of the samespecies as the tumor-bearing animal to permit biofiltration of theantibodies;

(c) recovering the biofiltered antibodies from each of thenon-tumor-bearing primate, the tumor-bearing animal and thenon-tumor-bearing animal;

(d) identifying antigens whose antibodies are not retained in thenon-tumor-bearing primate and in the non-tumor-bearing animal using therecovered biofiltered antibodies; and

(e) comparing the antibodies that are not retained in vivo by thenon-tumor-bearing primate and the non-tumor-bearing animal with thoseantibodies that are actually retained in the tumor-bearing animal toidentify the antigens corresponding to those antibodies that areselectively retained in the tumor-bearing animal.

The invention is based on the known extensive antigeniccross-reactivities among homologous proteins of higher primates. Becauseof this antigenic homology, it is possible to prepare antibodies againsthuman tumors, then test for absorption in primates (e.g., rhesusmonkeys, chimpanzees, orangutans). The antibodies are injected into theprimate, then blood is harvested after an appropriate interval. Serum orplasma is prepared from the blood samples, and the antibodyspecificities that are absorbed by the primate are determined by anappropriate method (e.g., "Western" immunoblotting). This stepdetermines those antigens whose antibodies remained unabsorbed in theprimate, and would therefore be putatively unabsorbed by humans.

That antigens exist in the primate in biochemical forms and inanatomical locations similar to those found in humans can bedemonstrated by immunoblotting using primate and human tissues, andstaining the immunoblots with antibodies before and after biofiltration.Immunomicroscopic staining of similar tissue sections from the primateand human, again using the biofiltered antibodies, would allowconfirmation of similar anatomical locations.

The invention permits selection of those antigens whose antibodies areunabsorbed in normal humans, but are absorbed by tumors. The inventionfurther relates to a process for the in vivo selection of antigenssuitable as targets for antibodies which localize in tumors in humanorgans involving the use of a tumor-bearing human organ. Since primatetumors will only rarely be available, tumors suitable for absorption mayinclude either human tumors that have been xenografted to nude mice orother animals, or tumors that have been removed from a patient in such away as to allow ex vivo perfusion. As an opportune example of the lattermethod, an entire tumor-bearing kidney is usually removed during surgeryfor kidney cancer. The tumor-bearing kidney may be mounted on a suitableperfusion apparatus (routinely used for kidney transplantation) andantibodies added to the perfusion solution, then the perfusate testedfor absorption of antigenic specificities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of one embodiment of the process of the inventionto identify antigens to human renal cell carcinoma.

FIG. 2 is a depiction of a second embodiment of the process of theinvention to identify antigens to human renal cell carcinoma.

FIG. 3 is a photograph of strips cut from a Western Blot of a wholeunbiofiltered antibody: (#1), antibody biofiltered through a rhesusmonkey (#2), antibody biofiltered through a nude mouse (#3) and antibodybiofiltered through a nude mouse bearing a human renal carcinoma (RCC)tumor (#4). The two arrows indicate antigens whose correspondingantibodies were unabsorbed by the rhesus monkey and the nude mouse, butwere absorbed by the nude mouse bearing the human RCC tumor.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment of the invention as depicted by FIG. 1, theselection of antigenic specificities in primate models is, preferably,performed by the following steps:

1. Antibodies, preferably xenogeneic antibodies distinguishable fromthose of the animals in which biofiltration is to be performed, areprepared to the tumor of interest by immunizing animals.

2. Antibody-containing fractions (immunoglobulins) are purified from thesera of the immunized animals.

3. The purified immunoglobulins in appropriate amounts are injected intothe circulation of a normal, non-tumor bearing primate, such as a rhesusmonkey, a normal non-tumor bearing animal, and a tumor-bearing animal ofthe same species as the non-tumor-bearing animal; any antibodies whichcan be absorbed ("biofiltered") will be removed in this step.

4. After a suitable time interval, preferably between 8 and 36 hours, asample of serum from each of the non-tumor bearing primate, thenon-tumor bearing animal, and the tumor-bearing animal injected in (3)is removed; these sera contain the "biofiltered" antibodies.

5. The biofiltered antibody is then used to identify biochemically theantigens corresponding to the biopurified antibodies by known methods.

The antigens of interest are those whose corresponding antibodies areunabsorbed by the primate and the non-tumor-bearing animal, but areabsorbed by the tumor-bearing animal.

Among the biochemical methods which may be used are gel blots,immunoprecipitation, and solid-phase adsorption of tumor substances.Preferably, gel blotting is used. These biochemical methods can be usedby employing a second antibody specific to antibodies from theoriginally immunized animal.

6. Confirmation that the antibodies do define a specificity suitable forin vivo targeting is done by (a) selecting specific antibodies definedby biofiltration, (b) radiolabeling the selected antibodies, then (c)injecting the radiolabeled, selected antibodies into tumor-bearinganimals.

7. The antigens defined by these biofiltered polyclonal antibodies maybe purified and then used as immunogens for preparation of monoclonalantibodies in accordance with known methods, such as the method ofDamsky et al., Cell 34, 355-366, 1983, the disclosure of which isincorporated herein by reference. An important feature in testing themonoclonal antibodies is competition with the polyclonal antibodiesisolated in steps 1-6 described above, thus ensuring that the monoclonalantibodies are directed to the same antigens as the biopurifiedpolyclonals.

In a further embodiment of the invention as depicted by FIG. 2, theselection of antigenic specificities is performed by the followingsteps:

1. Antibodies, preferably xenogenic antibodies, distinguishable fromthose of the animals in which biofiltration is to be performed anddistinguishable from components of the perfusion fluids to be used, areprepared by immunizing animals.

2. Antibody-containing fractions (immunoglobulins) are purified from thesera of the immunized animals.

3. The purified immunoglobulins in appropriate amounts are injected intothe circulation of a normal, non-tumor-bearing primate, such as a rhesusmonkey, and into the perfusion fluid of a human tumor that has beensurgically removed so as to allow perfusion of the tumor (for example, awhole kidney is normally removed when the kidney bears a renal cellcarcinoma; the removed kidney may be mounted in a perfusion apparatus ofthe sort normally used for preserving kidneys for transplantation).

4. After a suitable interval (preferably 8 to 36 hours for the primate,and 2 to 36 hours for the perfused organ, depending on perfusion volume,flow rate, and the preservation state of the organ), a sample of serumfrom the primate and a sample of the perfusion fluid from the perfusedtumor are removed. These samples contain the "biofiltered" antibodies.

5. The biofiltered antibody is then used to identify biochemically theantigens corresponding to the biopurified antibodies by known methods.The antigens of interest are those whose corresponding antibodies areunabsorbed by the primate, but are absorbed by the tumor-bearing organ.In the most preferred technique, a normal organ corresponding to thetumor-bearing organ (e.g. from a cadaver) will also have been perfusedin a fashion identical to the tumor-bearing organ. In that case,antigens of interest will be those whose corresponding antibodies areunabsorbed by the primate and by the normal organ, but are absorbed bythe tumor-bearing organ.

Among the biochemical methods which may be used are gel blots,immunoprecipitation, and solid-phase adsorption of tumor substances.Preferably, gel blotting is used. These biochemical methods can be usedby employing a second antibody specific to antibodies from theoriginally immunized animal.

The present invention provides for at least three additionalpossibilities for selection of specificities suitable for tumortargeting in humans:

1. Antigen-binding fragments could be prepared from the initial wholepolyclonal antibodies, then biopurification could be performed in humanvolunteers. The rationale is that, since such fragments lack effectorregions, any chance of cell- or complement-mediated damage to humanswill be avoided. Purification of antigens and preparation of monoclonalantibodies would follow as described above.

2. Prospective organ donors which cannot be used for transplantation(e.g., because of infection) could be used for biofiltration, giveninformed consent by next of kin.

3. Targetable human antigens may be defined by homology to targetableanimal antigens. Earlier work of the inventor has shown that biofilteredantibodies to mouse tumor antigens cross-react with antigens derivedfrom human tumors. A number of these human antigens are biochemicallysimilar to those found in mice, and hence are probably true homologues.These antigens should, therefore, be similarly targetable in humantumors, but not in normal tissues.

EXAMPLES Experimental Procedure

Example: Selection of antigens suitable as targets for antibodies whichlocalize in human renal cell carcinoma (RCC) (kidney cancer) tumors

1. Immunize rabbits using human RCC extract.

2. Prepare antisera from rabbits; purify immunoglobulin fraction bycolumn chromatography.

3. Re-inject rabbit immunoglobulins into normal primate, normal animaland animal of the same species bearing a human tumor. Antibodies whichbind to normal tissues and to the tumor are absorbed.

4. After a suitable time interval, preferably 24 to 72 hours, removeserum sample from normal primate, normal animal, and tumor-bearinganimal; these samples contain rabbit immunoglobulins that were notabsorbed by the normal primate and normal animal and that were absorbedby the tumor.

The three serum samples are compared by standard "Western"immunoblotting. When the strips stained by rabbit antibody beforebiofiltration were compared with those stained with an antibodybiofiltered (preferably after 8-36 hours) through a primate, anon-tumor-bearing animal, or a tumor-bearing animal, fewer antigens weredetected using the biofiltered than the input antibody. The "missing"antibody specificities are those retained by the primate or the otheranimals.

6. The antibodies which remain after biofiltration through the primateand through the normal animal, but not through the tumor-bearing animal,define antigens suitable for tumor targeting in humans. To show thatthese antibodies actually will target a tumor, the antigens arepurified, then used to prepare monospecific antibodies by affinitychromotagraphy. These selected antibodies are radiolabeled and injectedinto normal and tumor-bearing animals or perfused through normal andtumor-bearing organs.

7. Those antibodies which concentrate in the tumors will define antigenssuitable as targets. Antigens corresponding to the antibodies whichsuccessfully targeted tumors may be used as immunogens for preparationof monoclonal antibodies.

It should also be possible to "bootstrap" purification of antigens by(a) purification of a small amount of antigen; (b) immunization usingthe purified antigen; and (c) repeat purification using the nowrelatively monospecific antibody.

Example 1

Rabbit antisera to human renal cell carcinoma (RCC) were developed byrepeated immunizations at biweekly intervals using the whole tumorhomogenized in saline (1 mg protein/immunization). All immunizationswere subcutaneous; the first using 1:1::tumor homogenate: Freund'scomplete adjuvant, while all subsequent immunizations were done usingthe same proportion of Freund's incomplete adjuvant. After six months,rabbits were bled and serum was fractionated by standard techniques. TheIgG fraction was isolated by gel filtration chromatography on SephacrylS-200 (Pharmacia).

The r Rabbit antibodies were passaged through a rhesus monkey byintraveneous injection; the animal was bled twelve hours later, andserum prepared from the whole blood. Antibody was also passagedsimilarly through a nude mouse, and through a nude mouse bearing a humankidney tumor. The recovered sera were diluted in 10% fetal calf serum inDulbecco's PBS, then incubated overnight on sections of a "Western blot"of a Laemmli SDS gel prepared using the human renal cell carcinoma.After overnight incubation, the blots were washed using 0.05% Tween 20in tris-buffered saline, then incubated with 1/500 peroxidase-conjugatedgoat antibody to rabbit IgG (obtained from Organon Technica) for threehours at room temperature. The blots were re-washed to remove unabsorbedantibody, then developed using 4-chloro-1-naphthol using the proceduresdescribed in Allen, R. C., Saravis, C. A., and Maurer, H. R. (1984) GelElectrophoresis and Isoelectric Focusing of Proteins. New York: Walterde Gruyter, pp. 222-225, the disclosure of which is incorporated hereinby reference.

FIG. 3 shows the results from this test using rabbit antibodiesdeveloped to a human renal cell carcinoma. All results are from anSDS-Western Blot of the original human kidney tumor. The figure showsfour strips from an immunoblot labeled using (1) whole antibody beforeinjection; (2) antibody after passage through rhesus monkey; (3)antibody after passage through a normal nude mouse; and (4) antibodyafter passage through a nude mouse xenografted with a human tumor. Thetwo arrows indicate two specificities whose antibodies were unabsorbedby monkeys and by a normal nude mouse, but were absorbed by a nude mousebearing a human tumor.

Although the invention has been described in detail for the purposes ofillustration, it is to be understood that such detail is solely for thatpurpose and that variations can be made therein by those skilled in theart without departing from the spirit and scope of the invention exceptas it may be limited by the claims.

What is claimed is:
 1. A process for the selection of antigens suitableas targets for antibodies which localize in tumors in humans in vivocomprising:(a) preparing antibodies that bind to antigens present in ahuman tumor; (b) injecting the prepared antibodies into anon-tumor-bearing primate and into the perfusion fluid of a perfusedhuman tumor; (c) recovering the biofiltered antibodies from each of thenon-tumor-bearing primate and the perfused human tumor; (d) identifyingantigens whose antibodies are not retained in vivo in thenon-tumor-bearing primate but are retained by the perfused human tumor;and (e) comparing the antibodies to determine which specificities arenot retained in vivo by the non-tumor-bearing primate, but are retainedin the perfused human tumor, to identify the antigens corresponding tothose antibodies retained only in the perfused human tumor.
 2. Theprocess of claim 1 wherein the screening in step (e) further comprisesselecting specific antibodies which survive the biopurification of step(b), radiolabeling the selected antibodies and injecting theradiolabeled selected antibodies into the human tumor.
 3. The process ofclaim 1 wherein antigens whose antibodies are not retained in thenon-tumor-bearing primate, but are retained in the perfused human tumor,are identified in step (d) by a biochemical method.
 4. The process ofclaim 3 wherein antigens whose antibodies are not retained in thenon-tumor-bearing primate, but are retained in the perfused human tumor,are identified in step (d) using a biochemical method selected from thegroup consisting of Western blots, immunoprecipitation and solid-phaseadsorption.
 5. The process of claim 1 wherein the antibodies prepared instep (a) are xenoantibodies.
 6. The process of claim 5 wherein theantibodies prepared in step (a) are polyclonal xenoantibodies.
 7. Theprocess of claim 5 further comprising:(f) preparing monoclonalantibodies using the antigen identified in step (e) as immunogens.